Project Summary/Abstract Only recently has the extent of the diversity of innate immune effector functions become apparent, with the discovery of multiple novel subtypes of innate lymphoid cells (ILCs), beyond long recognized natural killer cells. ILCs derive from common lymphoid progenitors (CLP) in the bone marrow, and a number of transcriptional regulators that are involved in ILC development also play roles in the development of T cells in the thymus. Among these is TOX (thymocyte-selection associated HMG-box protein), a nuclear DNA binding protein and member of the HMG-box superfamily of proteins. Our previous work has shown that TOX is required for CD4 T cell lineage development in the thymus, and for early ILC lineage specification. Using a TOX reporter strain of mouse we have now identified bone marrow progenitor cells that may be in transition from CLP to the common progenitor to all helper-like innate lymphoid cells (CHILP). CHILP highly express the transcriptional regulator Id2, but we have identified Tox+Id2lo putative precursor cells, suggesting that upregulation of TOX likely precedes the formation of CHILP. We propose to test the cell fate potential of these cells in vitro and in vivo, to determine what ILC lineages they can form and if they have lost T cell potential. Similarly, we will ask if ILC lineage specification even precedes Tox upregulation. Armed with this knowledge, we then propose to determine the transcriptome in these cell types, at the single cell level, and to determine the influence of TOX on gene expression. Notch signaling is also thought to play a role in early ILC lineage specification. We propose that transient Notch signaling is key to distinguish ILC from T cell lineage commitment. Using an in vitro model system that allows differentiation of CLP to multiple ILC subtypes, we will study the cell fate and transcriptional consequences of a temporally limited Notch signal. As Tcf7 (encoding TCF-1), a key Notch signaling target gene, is likely downstream of TOX, we ask if expression of Tcf7 can compensate for loss of TOX. Finally, we address the underlying mechanism of action of TOX, using in vitro and in vivo approaches to ask if TOX functions as part of a novel RSF ISWI chromatin-remodeling complex, and propose to produce a novel mouse strain to aid in identification of gene targets and protein binding partners of TOX in multiple cell contexts.